A HISTOLOGICAL PROCESSING TECHNIQUE THAT PRESERVES THE INTEGRITY OF CALCIFIED TISSUES (BONE, ENAMEL), YOLKY AMPHIBIAN EMBRYOS, AND GROWTH-FACTOR ANTIGENS IN SKELETAL TISSUE

We have devised a processing technique to embed calcified tissues, suc h as bone and tooth enamel, in paraffin, to preserve the delicate anti genic sites of molecules such as growth factors. The same technique, o mitting the decalcification step, allows delicate tissues, such as axo lotl embryos (Ambystoma mexicanum) containing large yolk masses, to be easily handled during tissue processing and to be serially sectioned. Specimens were all fixed in periodate-lysine-paraformaldehyde (PLP) f ixative at 5-degrees-C. Bone and teeth were decalcified in an EDTA-G s olution at -4-degrees-C. Maintaining a temperature of 5-degrees-C, the decalcified samples were then washed (with PBS, pH 7.2, under vacuum) to remove glycerol. Both the decalcified tissues and the yolky embryo s were dehydrated through an ascending series of isopropanol and embed ded in low melting-point paraffin under vacuum. Acidic fibroblast grow th factor (aFGF) was located in cells of the expanded cambial layer in the early fracture calluses of male CD-1 mice, demonstrating retentio n of antigenic sites. The results reported here have not previously be en obtained with existing processing and embedding techniques.